Neutralization of IL-4 and IFN-γ Facilitates inducing TGF-β-induced CD4(+)Foxp3(+) Regulatory Cells.

It has been well recognized that TGF-β is able to induce CD4(+)CD25(+)Foxp3(+) suppressor/regulatory T (iTreg) cells and IL-2 facilitates iTreg induction and expansion, however, only half of TGF-β-induced CD4(+)CD25(+) cells express Foxp3 and remaining CD4(+)CD25(+)Foxp3- cells may represent effector cells. Whether other factor(s) can increase Foxp3 expression by CD4(+)CD25(+) cells induced with TGF-β is still unclear. Here we show that addition of exogenous IFN-γ or IL-4 diminished the ability of TGF-β to induce Foxp3 expression and IL-2 failed to rescue this decreased Foxp3 expression. Conversely, neutralization of IFN-γ and IL-4 significantly enhanced the ability of TGF-β to induce Foxp3 and develop the suppressive activity, indicating that different cytokine profiles affect the differentiation of CD4(+)CD25(+)Foxp3(+) subset induced by TGF-β. These results show that combination of antibodies against IFN-γ and IL-4 and TGF-β enhances the efficacy of generation and function of iTreg cells and may therefore provide a novel therapeutic strategy for the treatment of autoimmune and other chronic inflammatory diseases.


INTroDuCTIoN
Naturally occurring, thymus-derived CD4 + CD25 + suppressor/regulator T cells (nTregs) play a pivotal role in maintenance of immune tolerance to self antigens (1,2). Lack or dysfunction of nTregs have appeared to be involved in the development and progression of autoimmune diseases, such as type I diabetes, multiple sclerosis, rheumatoid arthritis and active lupus (3)(4)(5)(6). nTregs account for only 5%-10% of peripheral CD4 + T cells in mice and 1-2% in human (7). Foxp3, one of forkhead family transcription factor members, is a lineage specification factor for Tregs and plays an important role in the development and function of Treg cells (8,9). It is likely that manipulation of nTreg cells may provide another approach to treat autoimmune diseases. Nonetheless, small numbers and decreased suppressive activity following expansion limit their potential for therapeutic considerations (10).
Treg cells are heterogeneous and composed of either thymus-derived nTregs or those that can be induced outside of the thymus (induced Treg, iTreg) (10). Although
TGF-β is a pleiotropic cytokine exerting a differential impact on the differentiation of T lymphocytes depending on the target cell type and distinct cytokine milieu (22). Whereas TGF-β induces the differentiation of Foxp3 + Treg cells in the presence of IL-2 (23,24), TGF-β also facilitates the induction of IL-17-producing (Th17) cells, at least in animal models (25)(26)(27). In addition, TGF-β has a critical function as an antagonist of Th1 development affecting IFN-γ as well as T-bet (28,29), of Th2 differentiation affecting IL-4 (30,31). Although it has been reported that non-T cell-derived IL-6 abolishes the ability of TGF-β to induce Foxp3 + cells (32), it is still unclear whether other Th1 and Th2 cytokines produced by T cells also affect the differentiation of iTreg cells induced by TGF-β.
In the present work, we confirmed that TGF-β is able to induce naïve CD4 + CD25cells to express Foxp3 and develop suppressive activity in the absence of antigen presenting cells (APC). However, the addition of exogenous IFN-γ or IL-4 diminished the ability of TGF-β to induce Foxp3 expression. Interestingly, neutralization of IFN-γ and IL-4 significantly enhanced the ability of TGF-β to induce Foxp3 and develop the suppressive activity. Thus, the combination of TGF-β and neutralization of IFN-γ and IL-4 may provide a new protocol for the generation of TGF-β-induced iTreg cells ex vivo.

Mice
C57BL/6 mice were purchased from The Jackson Laboratory and Shanghai Animal Institute, respectively. Mice used in all experiments were 8-12 weeks of age. All animals were treated according to National Institutes of Health guidelines for the use of experimental animals with the approval of the University of Southern California Committee for the Use and Care of Animals and Natural Science Foundation of China guidelines for the use of animals with approval of Committee for the Use and Care of Animals from Zhejiang Traditional Chinese Medicine and Western Medicine Hospital, P. R. China.

Cell Purification
T cells were prepared from spleen cells by collecting nylon wool column non-adherent cells as described previously (17). CD4 + T cells were isolated by magnetic beadbased negative selection. In brief, T cells were labeled with PE-conjugated anti-CD8, anti-CD11b, and anti-B220 mAbs, incubated with anti-PE magnetic beads, and loaded onto MACS separation columns (Miltenyi Biotec). CD4 + cells were further labeled with FITC-conjugated anti-CD25 mAb, and CD4 + CD25 + and CD4 + CD25cells were obtained by cell sorting (cell purity of CD4+CD25-cells is >98%). To prepare naïve CD4 + CD25cells, CD4 + CD25cells were labeled with PE-conjugated anti-CD62L and positively selected by anti-PE magnetic beads.

Flow cytometry analysis and intracellular cytokine staining
Prior to staining, cells were washed and re-suspended in staining buffer containing 1x PBS, 2% BSA, 10mM EDTA and 0.01% NaN3, To block non-specific staining, anti-CD16/32 antibody (2.4G2) was added. Antibodies for cell surface markers were added and cells were incubated 25 min on ice. Following staining, the cells were washed twice and analyzed the same day or fixed in PBS containing 1% paraformaldehyde and 0.01% NaN3, and cells were examined on the Epics XL-MC and data analyzed using EXPO32 software. Intracellular Foxp3 staining was performed as per Foxp3-staining kit protocol.

In vitro proliferation/suppression assays
Proliferation assays were performed by stimulating responding T cells in 96 flat-bottom microtiter plates in RPMI 1640 with immobilized anti-CD3 (0.5 µg/ml), soluble anti-CD28 for 72 h at 37°C in 5% CO 2 . For suppression assays, TGF-beta1-treated or untreated T cells were cocultured with CD4 + CD25-responder T cells with immobilized anti-CD3 (0.5 µg/ml), soluble anti-CD28 in 96-well plates for 72 h at 37°C/5% CO2. Cell cultures were pulsed with 1 uCi 3 H-thymidine for the last 16 h to determine the extent of suppression.

rT-PCr for Foxp3 expression
Total RNA was extracted from cells using TRIzol reagent and used to determine the expression and relative level of the transcription factor Foxp3. First-strand cDNA was synthesized using Omniscript TR kit (Qiagen) with random hexamer primers (Invitrogen Life Technologies). Foxp3 and hypoxanthine guanine phosphoribosyl transferase (HPRT) mRNA was measured by a semiquantitative RT-PCR using published primers (8). The relative expression of Foxp3 was determined by normalizing expression of each target to HPRT.

statistical analysis
Results are expressed as mean ± SEM, and are representative of 3-5 similar experiments. Analysis for statistically significant differences was performed with Student's t-test. P<0.05 was considered a difference, and P<0.01 was considered a significant difference.

resuLTs addition of exogenous IFN-γ or IL-4 decreases the Foxp3 expression induced by TGF-β
As described previously by us and others, naïve CD4 + CD25-T cells activated with anti-CD3/CD28 in the presence IL-2 and TGF-β become CD4 + CD25 + and more than 50% CD25 + cells have been converted into Foxp3 + cells (13, 23, Fig.1). TGF-β plays a unique role in the induction of Foxp3 expression and development of suppressive activity since TGF-β failed to induce CD4 + CD25cells from TGF-β receptor II dominate mice to express Foxp3 (data not shown). As IL-2 facilitates the induction of Foxp3 expression and IL-6 diminishes Foxp3 expression induced by TGF-β (20,21,32), it is possible other cytokines also affect the Foxp3 expression in CD4 + CD25 + cells induced by TGF-β. We consider the possibility that cytokines which induce the differentiation of Th1 or Th2 cells may reduce the ability of TGF-β to induce Foxp3 since TGF-β suppresses Th1 and Th2 differentiation. As shown in Fig. 1, addition of exogenous IFN-γ or IL-4 markedly decreased the ability of TGF-β to induce Foxp3. These data indicate that different cytokines affect the differentiation of CD4 + cells and induction of TGF-β-iTreg cells needs specific cytokine profiles in addition to TCR engagement.
We also assessed suppressive activities of CD4 + CD25 + cells induced by IL-2 and TGF-β with or without anti-IFN-γ and anti-IL-4 or control IgG. The experiment shown in Fig. 3 reveals that the suppressive activity of iTreg induced by IL-2 and TGF-β in the presence of both anti-IFN-γ and anti-IL-4 antibodies is significantly greater than in the absence of antibodies. These data suggest that neutralization of IFN-γ and IL-4 not only increases the Foxp3 expression in CD4 + CD25 + cells induced by IL-2 and TGF-β, but also enhances the suppressive activity of these cells.

DIsCussIoN
The present study evaluates the ability of neutralization of IFN-γ and IL-4 to induce and enhance suppressive func-tions of TGF-β-induced CD4 + CD25 + cells. We show that TCR engagement in conjunction with TGF-β stimulation can up-regulate Foxp3 expression and induce the suppressive activity of iTreg cells from peripheral CD4 + CD25 -T cell progenitors. Whereas we demonstrate that exogenous IFN-γ or IL-4 diminishes the ability of TGF-β to induce CD4 + CD25cells to express Foxp3, we also observed that the neutralization of IFN-γ and IL-4 markedly promote Foxp3 expression by TGF-β-induced iTreg cells and significantly enhances the suppressive activity of these cells.
Although TGF-β suppresses the differentiation of Th1 and Th2 cells, the existence of IFN-γ or IL-4, which respectively promotes the differentiation of Th1 or Th2 cells (33), interrupts the differentiation of Foxp3 + Treg cells ini-A Figure  1. Exogenous IFN-γ or IL-4 diminishes the ability of TGF-β to induce CD4 + CD25cells to express Foxp3. Splenic naïve CD4 + CD25cells isolated from C57BL/6 mice were stimulated with immobilized anti-CD3, soluble anti-CD28 and IL-2 ± TGF-β with or without exogenous IFN-γ or IL-4 for four days, and examined by flow cytometry.  Conditioned CD4 + cells were similarly generated as described in Figure 2. Suppression was assayed using anti-CD3stimulated cells as described in Materials and Methods. The ratios of CD4 med (without TGF-β) or CD4 TGF-β to responder T cells are shown (1:8). This result is representative of four independent experiments. NIL indicates no added cells. P values indicate significant effects of anti-IFN-γ and anti-IL-4 on TGF-β treated cells compared to control IgG (p=0.02). tiated by TGF-β. It is similar that addition of IFN-γ or IL-4 inhibits the differentiation of Th17 cells induced by combination of IL-6 and TGF-β (25)(26)(27). IL-2 fails to overcome the effect of IFN-γ or IL-4 on TGF-β-induced Foxp3 expression in CD4 + cells although IL-2 critically involves in the development and expansion of TGF-β-induced Foxp3 + Tregs (23,24).
The mechanism(s) by which IFN-γ or IL-4 affects the ability of TGF-β to induce Fopx3 + Treg cells remains unclear. Although cytokines promoting the differentiation of Th1, Th2, Th17 or Treg cells are known to antagonize each other (34), it is also possible that they have some synergizing role in promoting the differentiation of distinct CD4 + subsets. Others have reported while IL-4 favors Th2 differentiation, combination of IL-4 and TGF-β resulted in generation of Th1 cells (35). The importance of IFN-γ in regulating TGF-β production was further confirmed in the study showing that CD4 + cells from IFN-γ -/mice produced more TGF-β compared to wild type mice (35).
It is likely the neutralization of IFN-γ or IL-4 enhances Foxp3 expression in CD4 + CD25 + cells induced by TGF-β. As shown in Fig. 1 and reported previously, only 50% of TGF-β-induced CD4 + CD25 + cells express Foxp3 (23). This implies that fully half of, the TGF-β-induced CD4 + CD25 + cells do not express Foxp3 and may represent CD4 + effector cells. In order to reliably generate induced Treg cells ex vivo, it will be important to increase CD25 + Foxp3 + and decrease CD25 + Foxp3cells since suppressive activity is closely associated with Foxp3 expression (23). This study establishes a new protocol that will improve the ability of TGF-β to induce iTreg cells ex vivo, thereby increasing the likelihood that manipulation of TGF-β-induced iTreg cells may provide a novel therapeutic strategy for the treatment of autoimmune diseases and other chronic inflammatory diseases.